Humic substances and the water calcium content change the toxicity of malachite green

Laboratory experiments were conducted to test interactive effects of calcium (Ca²⁺) content and the presence of humic substance (HS) on malachite green (MAG)‐induced toxicity in fish embryos and larvae by means of a semistatic 144‐h‐embryo‐larval‐test with zebrafish (Danio rerio). Two kinds of reconstituted water samples were used to produce the test media by mixing salts into deionized water resulting in either hard water (↑Ca ? HS), or soft water (↓Ca ? HS). By adding HS two additional test media were produced (↑Ca + HS, ↓Ca + HS). MAG was tested in concentrations of 0.05, 0.10, 0.15, 0.20, 0.25 mg L^?1. The toxicity ranking of MAG (mg L^?1) to embryos based on 96‐h‐LC₅₀ in the different test water samples is: ↑Ca ? HS (0.061) > ↑Ca + HS (0.123) = ↓Ca ? HS (0.12) ≥ ↓Ca + HS (0.134) and on 144‐h‐LC₅₀ to larvae is: ↑Ca ? HS (0.038) > ↑Ca + HS (0.06) > ↓Ca ? HS (0.077) = ↓Ca + HS (0.077). Mortality of all the groups was significantly different (P < 0.05). Increased Ca²⁺ concentrations did not protect zebrafish embryos and larvae from MAG‐induced toxicity. At high Ca²⁺ conditions, the mortality of the embryos as well as of the larvae is reduced in the ↑Ca + HS group relative to the ↑Ca ? HS group. Thus, at high Ca²⁺ conditions the HS does affect the MAG‐induced mortality. The mechanism which causes the higher toxicity of MAG in the presence of higher Ca²⁺ concentrations is poorly understood. A probable explanation could be the stimulation of the calcium‐binding protein calmodulin as well as the calmodulin kinase II in cell membranes in the presence of high Ca²⁺ concentrations.     

Cytotoxic effects of methanol, formaldehyde, and formate on dissociated rat thymocytes: A possibility of aspartame toxicity

Aspartame is a widely used artificial sweetener added to many soft beverages and its usage is increasing in health-conscious societies. Upon ingestion, this artificial sweetener produces methanol as a metabolite. In order to examine the possibility of aspartame toxicity, the effects of methanol and its metabolites (formaldehyde and formate) on dissociated rat thymocytes were studied by flow cytometry. While methanol and formate did not affect cell viability in the physiological pH range, formaldehyde at 1–3 mmol/L started to induce cell death. Further increase in formaldehyde concentration produced a dose-dependent decrease in cell viability. Formaldehyde at 1 mmol/L or more greatly reduced cellular content of glutathione, possibly increasing cell vulnerability to oxidative stress. Furthermore, formaldehyde at 3 mmol/L or more significantly increased intracellular concentration of Ca²⁺([Ca²⁺]_i) in a dose-dependent manner. Threshold concentrations of formaldehyde, a metabolite of methanol, that affected the [Ca²⁺]_iand cellular glutathione content were slightly higher than the blood concentrations of methanol previously reported in subjects administered abuse doses of aspartame. It is suggested that aspartame at abuse doses is harmless to humans. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of environmental calcium deprivation on the egg masses of Physa marmorata Guilding (Gastropoda: Physidae) and Biomphalaria glabrata Say (Gastropoda: Planorbidae)

Eggs of the basommatophoran snails Physa marmorata and Biomphalaria glabrata were cultured in low concentrations of calcium to determine effects on growth and development. In both species there was some development in media with 0.12 mg/l Ca²⁺ but embryos were unable to hatch. 61.04% of embryos of P. marmorata could develop to hatching in 0.22 mg/l Ca²⁺ but those of B. glabrata required a level of 0.42 mg/l Ca²⁺, to attain even a 31.07% hatch. Marked effects on growth rate, embryo size and on time taken to achieve hatching were noted in both species at very low calcium levels. The possibility of cation-controlling mechanisms in the egg membrane is discussed.     

Calcium-dependent ATPase unlike ecto-ATPase is located primarily on the luminal surface of brain endothelial cells

Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca²⁺-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca²⁺-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca²⁺ was replaced by Mg²⁺ or Mn²⁺ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca²⁺-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca²⁺-specific-ATPase luminal localization explains the stable Ca²⁺ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.     

Peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line

It has long been concerned that some secondary air pollutants such as smog components, ozone (O₃) and peroxyacetyl nitrate (PAN), are highly phytotoxic even at low concentrations. Compared with the biology of O₃, we largely lack the information on the toxicity model for PAN at the cellular signaling levels. Here, we studied the cell-damaging impact of PAN using suspension culture of smog-sensitive tobacco variety (Bel-W3). The cells were exposed to freshly synthesized PAN and the induced cell death was assessed under microscope after staining with Evans blue. Involvement of reactive oxygen species (ROS) in PAN toxicity was suggested by PAN-dependently increased intracellular H₂O₂ and also by the cell-protective effects of ROS scavengers and related inhibitors. Calcium chelator also lowered the level of PAN-induced cell death, indicating that Ca²⁺ is also involved. Using a transgenic cell line expressing aequorin, an increase in cytosolic Ca²⁺ concentration responsive to the pulse of PAN, but sensitive to Ca²⁺ channel blockers, was recorded, indicating that Ca²⁺ channels are activated by PAN or PAN-derived signals. Above data show some similarity between the signaling mechanisms responsive to O₃ and PAN.     

Aluminum effects on calcium fluxes at the root apex of aluminum-tolerant and aluminum-sensitive wheat cultivars

The role of Ca²⁺ transport in the mechanism of Al toxicity was investigated, using a Ca²⁺-selective microelectrode system to study Al effects on root apical Ca²⁺ fluxes in two wheat (Triticum aestivum L.) cultivars: Al-tolerant Atlas 66 and Al-sensitive Scout 66. Intact 3-day-old low-salt-grown (100 micromolar CaCl₂, pH 4.5) wheat seedlings were used, and it was found that both cultivars maintained similar rates of net Ca²⁺ uptake in the absence of Al. Addition of Al concentrations that were toxic to Scout (5-20 micromolar AlCl₃) immediately and dramatically inhibited Ca²⁺ uptake in Scout, whereas Ca²⁺ transport in Atlas was relatively unaffected. The Al-induced inhibition of Ca²⁺ uptake in Scout 66 was rapidly reversed following removal of Al from the solution bathing the roots. Similar studies with morphologically intact root cell wall preparations indicated that the Al effects did not involve Al-Ca interactions in the cell wall. These results suggest that Al inhibits Ca²⁺ influx across the root plasmalemma, possibly via blockage of calcium channels. The differential effect of Al on Ca²⁺ transport in Al-sensitive Scout and Al-tolerant Atlas suggests that Al blockage of Ca²⁺ channels could play a role in the cellular mechanism of Al toxicity in higher plants.     

Calcium mediated cytokinin action on chlorophyll synthesis in isolated embryo of Scots pine

Chlorophyll (Chl) synthesis in isolated Scots pine embryos depended on exogenous application of cytokinin (CK) and Ca²⁺. At a constant benzyladenine (BA) level (4.4×10^?5 M) 10^?4 to 10^?2 M Ca²⁺ concentrations in mineral medium were optimum for Chl biosynthesis under both light and dark. At a zero or very low (10^?6 M) concentration of external Ca²⁺, Chl synthesis was relatively more Ca²⁺-dependent in embryos cultured in darkness than in the light, which suggested that the light: (a) stimulated the transport of Ca²⁺ from external sources to cytosol, and/or (b) interacted with Ca²⁺ directly in the pathway of Chl biosynthesis. The need of external Ca²⁺ was evidenced in experiments with modulators of Ca²⁺-transport systems. The reduction of the inward current of Ca²⁺ from readily accessible external sites by chelating agent (ethylene glycol-bis (beta-aminoethyl ether-N,N,N′N′-tetraacetic acid, EGTA) and Ca²⁺-channel blockers canceled the formation of Chl. The effect of EGTA depended on the level of external Ca²⁺. Inhibitory action of Ca²⁺-channel blockers depended on their kind and concentration: at the 10^?5 M concentration La³⁺>verapamil>nifedipine inhibited Chl formation. In the presence of Ca²⁺, the Ca²⁺-agonist A 23187 mimicked the BA effect and about 92% of Chl was synthesized as compared with the BA variant. Low concentrations of calmodulin antagonists reduced the amounts of Chl. Calmodulin was included in a second messenger system for BA action in promoting Chl biosynthesis in isolated Scots pine embryos.     

Cellular responses of developing Fucus serratus embryos exposed to elevated concentrations of Cu2+

Elevated concentrations of Cu²⁺ can have inhibitory effects on early development in plants and algae by targeting specific cellular processes. In the present study the effects of elevated Cu²⁺ on developmental processes in embryos of the brown algae Fucus serratus (Phaeophyceae) were investigated. Elevated Cu²⁺ was shown to inhibit fixation of the zygotic polar axis but not its formation. Actin localization was unaffected by elevated Cu²⁺ but polarized secretion, which occurs downstream, was inhibited. Significant differences in tolerance to Cu²⁺ were observed for polarization and rhizoid elongation of embryos derived from adults from Cu²⁺‐contaminated and uncontaminated locations. Moderate Cu²⁺ exposure inhibited the generation of cytosolic Ca²⁺ signals in response to hypo‐osmotic shocks. In contrast, cytosolic Ca²⁺ was elevated by treatments with high [Cu²⁺] and this coincided with production of reactive oxygen species. The results indicate that direct effects on signalling processes involved in polarization and growth may in part explain complex, concentration‐dependent effects of Cu²⁺ on early development.     

Stabilization of calcium uptake in rat rod outer segments by taurine and ATP

Summary. Calcium ion (Ca²⁺) uptake was measured in rod outer segments (ROS) isolated from rat retina in the presence of varying concentrations of CaCl₂ in the incubation buffer (1.0–2.5 mM). It is known that taurine increases Ca²⁺ uptake in rat ROS in the presence of ATP and at low concentrations of CaCl₂ (Lombardini, 1985a); taurine produces no significant effects when CaCl₂ concentrations are increased to 1.0 and 2.5 mM. With the removal of both taurine and ATP, Ca²⁺ uptake in rat ROS increased significantly in the presence of 2.5 mM CaCl₂. Taurine treatment in the absence of ATP was effective in decreasing Ca²⁺ uptake at the higher levels of CaCl₂ (2.0 and 2.5 mM). Similar effects were observed with ATP treatment. The data suggest that taurine and ATP, alone or in combination, limit the capacity of the rat ROS to take up Ca²⁺ to the extent that a stable uptake level is achieved under conditions of increasing extracellular Ca²⁺, indicating a protective role for both agents against calcium toxicity. Received January 25, 2000/Accepted January 31, 2000     

The Effect of Combined Exposure of 900 MHz Radiofrequency Fields and Doxorubicin in HL-60 Cells

Human promyelocytic leukemia HL-60 cells were pre-exposed to non-ionizing 900 MHz radiofrequency fields (RF) at 12 µW/cm² power density for 1 hour/day for 3 days and then treated with a chemotherapeutic drug, doxorubicin (DOX, 0.125 mg/L). Several end-points related to toxicity, viz., viability, apoptosis, mitochondrial membrane potential (MMP), intracellular free calcium (Ca²⁺) and Ca²⁺-Mg²⁺ -ATPase activity were measured. The results obtained in un-exposed and sham-exposed control cells were compared with those exposed to RF alone, DOX alone and RF+DOX. The results indicated no significant differences between un-exposed, sham-exposed control cells and those exposed to RF alone while treatment with DOX alone showed a significant decrease in viability, increased apoptosis, decreased MMP, increased Ca²⁺ and decreased Ca²⁺-Mg^2+-ATPase activity. When the latter results were compared with cells exposed RF+DOX, the data showed increased cell proliferation, decreased apoptosis, increased MMP, decreased Ca²⁺ and increased Ca²⁺-Mg²⁺-ATPase activity. Thus, RF pre-exposure appear to protect the HL-60 cells from the toxic effects of subsequent treatment with DOX. These observations were similar to our earlier data which suggested that pre-exposure of mice to 900 MHz RF at 120 µW/cm² power density for 1 hours/day for 14 days had a protective effect in hematopoietic tissue damage induced by subsequent gamma-irradiation.     

Interactions among Ca2+, Na+ and K+ in salinity toxicity: quantitative resolution of multiple toxic and ameliorative effects

Root elongation by wheat seedlings (Triticum aestivum L. cv. Scout 66) was not inhibited by NaCl or KCl up to 130 mM in culture solutions or by high Na⁺ (2 mg g^-1 FW) or K⁺ (4 mg g^-1 FW) in the root tissue, provided that [Ca²⁺]>2 mM in the rooting medium. At [NaCl], [KCl], or [mannitol] >250 mOs, root elongation was progressively inhibited, irrespective of high [Ca²⁺]. In contrast, shoot elongation was sensitive to any diminution of water potential, and Ca²⁺ alleviated the toxicity only weakly. At solute concentrations <250 mOs, the following interactions were observed. Ca²⁺ alleviated Na⁺ and K⁺ toxicity to roots by at least three separate mechanisms. K⁺ was more toxic to roots than Na⁺, but Na⁺ was more toxic to shoots. Low levels of K⁺ relieved Na⁺ toxicity, but low levels of Na⁺ enhanced K⁺ toxicity. Tissue concentrations of Na⁺ were reduced by Ca²⁺ and K⁺ in the rooting medium, and tissue concentrations of K⁺ were enhanced by Ca²⁺ and Na⁺. Several hypotheses relating to salinity toxicity can be evaluated, at least for wheat seedlings. The osmoticant hypotheses (salinity intoxication occurs because of diminished water potential) is true for shoots at all salinity levels, but is true for roots only at high salinity. The Ca²⁺-displacement hypothesis (Na⁺ is toxic because it displaced Ca²⁺ from the cell surface) is correct, but often of minor importance. The K⁺-depletion hypothesis (Na⁺ is toxic because it causes a loss of K⁺ from plant tissues) is false. The Cl^--toxicity hypothesis (the apparent toxicity of Na⁺ is induced by associated Cl^-) is false. The results indicate that, apart from osmotic effects, high levels of Na⁺ in the rooting medium and in the tissues are not toxic unless Ca²⁺ is also deficient, a condition probably leading to inadequate compartmentation and excessive cytoplasmic accumulation. This study related growth to ion activities at plasma-membrane surfaces. These activities were computed by a Gouy-Chapman-Stern model then incorporated into non-linear growth models for growth versus toxicants and ameliorants.Key words: Calcium, potassium, salinity, sodium, toxicity      

External calcium is required for activation of phospholipase C by angiotensin II in adrenal glomerulosa cells

Previous studies have shown that external calcium (Ca²⁺) is required for the effects of angiotensin II (AII) on aldosterone secretion in adrenal glomerulosa zone. Using bovine adrenal glomerulosa cells prepared by collagenase dispersion, we examined whether external Ca²⁺ is required for the activation of phospholipase C by AII. Adrenal glomerulosa cells were exposed to Ca-EGTA buffered media to provide accurate estimates of external free Ca²⁺ concentrations. Phospholipase C activation was evaluated by measurement of inositol phosphates production. At 0.1 M Ca²⁺ and less, sustained AII effects on inositol monophosphate (IP), inositol bisphosphate (IP₂) and inositol trisphosphate (IP₃) were markedly inhibited. Increasing the Ca²⁺ concentration to 50kM or greater fully restored All-induced inositol phosphates production. AII-induced increases in cytosolic Ca²⁺ measured by Quin-2 fluorescence, were diminished at lower external Ca²⁺ concentrations. Treating adrenal glomerulosa cells with Chelex-100, a strong Ca²⁺ binding resin, blocked early activation of phospholipase C by AII. Inhibition of IP₃ production was also observed when inhibitors of Ca²⁺ movement across the plasma membrane were used, viz., La²⁺, TMB-8 and nifedipine. The requirement for Ca²⁺ during AII-induced activation of phospholipase C may be explained, at least partly by a requirement for Ca²⁺ at a site between the AII receptor and Phospholipase C.     

Interactive effects of Ca2+ and NaCl salinity on the growth of two tomato genotypes differing in Ca2+ use efficiency

Two tomato (Lycopersicon esculentum Mill.) lines differing in Ca²⁺ use efficiency (Ca²⁺ use efficient line 113 and Ca²⁺ use inefficient line 67) were subjected to salinity treatments in two separate experiments to determine whether they differed in salt tolerance. In experiment I, three NaCl and two CaCl₂ treatments were imposed. The Na⁺ concentrations were 1.1, 100 and 150 mM and the Ca²⁺ concentrations were either 1.51 or 10 mM. In experiment II, one NaCl and three Ca²⁺ treatments (as CaCl₂ or CaSO₄) were imposed. The treatments consisted of 150 mM NaCl at either 1.51 mM CaCl₂, 10 mM CaCl₂, or 10 mM CaSO₄. Response to treatments was determined by analysis of growth parameters (shoot and root dry weights, plant height, and root length). Shoot and root dry weight, and root length were depressed as salinity increased in plants lacking additional Ca²⁺. No significant differences in salt tolerance were detected between the two tomato lines after 24 d of salinity treatment. An important finding of this study was that root growth and length appeared to be more sensitive to the effect of CaCI₂ treatment alone and to the effects of CaCl₂ × NaCl treatments. This suggests that over the long term, both root growth and root length may be more sensitive indicators of salinity effects than shoots. Supplemental CaCl₂ had no ameliorative effect on NaCl stress in shoot growth. The inability of Ca²⁺ to counter Cl⁻ entry or toxicity may account for the lack of amelioration. Additional Ca²⁺ as CaSO₄ improved shoot growth of plants exposed to 150 mM NaCl. In contrast, root growth and length were improved by 10 mM Ca²⁺ as either CaCl₂ or CaSO₄.     

Synergistic action of calcium-lonophores and hyperthermia is best interpreted as thermal enhancement of calcium toxicity

It has been shown that no relation exists between [Ca²⁺]_i and hyperthermic cell killing, although heat-induced increase of [Ca²⁺]_i can be observed in some cell lines. When ionophores are used, dose-dependent rises in [Ca²⁺]_i may be found. Beyond a certain threshold of ionophore-induced increases in [Ca²⁺]_i, cells may be killed. Different threshold levels of [Ca²⁺]_i exist in different cell lines. Hyperthermia can act synergistically with calcium ionophores to potentiate cell killing. Since there is no causal relation between [Ca²⁺]_i and heat toxicity, this synergism can be explained as heat enhanced Ca²⁺ toxicity. In the current report, it is shown that both ionophore-induced Ca²⁺ toxicity (37°C) and its potentiation by heat are dependent on extracellular calcium and related to sustained increases in [Ca²⁺]_i. With ionomycin concentrations up to 15 μM, no increase in [Ca²⁺]_i was seen in cells maintained in medium without Ca²⁺. Ionomycin effects on intracellular compartments were absent, and the drug seemed to act solely on the level of the plasmamembrane. Also, the synergism of heat and ionomycin appeared to act at the plasmamembrane, because depletion of extracellular calcium completely abolished this synergistic effect. The data presented are also discussed in the light of controversies existing in the literature for the role of calcium in hyperthermic cell killing. © 1993 Wiley-Liss, Inc.     

Cadmium inhibits plasma membrane calcium transport

Summary The interaction of Cd²⁺ with the plasma membrane Ca²⁺-transporting ATPase of fish gills was studied. ATP-driven Ca²⁺-transport in basolateral membrane (BLM) vesicles was inhibited by Cd²⁺ with anI ₅₀ value of 3.0nm at 0.25 m free Ca²⁺ using EGTA, HEEDTA and NTA to buffer Ca²⁺ and Cd²⁺ concentrations. The inhibition was competitive in nature since theK _0.5 value for Ca²⁺ increased linearly with increasing Cd²⁺ concentrations while theV _max remained unchanged. The Ca²⁺ pump appeared to be calmodulin dependent, but we conclude that the inhibition by Cd²⁺ occurs directly on the Ca²⁺ binding site of the Ca²⁺-transporting ATPase and not via the Ca²⁺-binding sites of calmodulin. It is suggested that Cd²⁺-induced inhibition of Ca²⁺-transporting enzymes is the primary effect in the Cd²⁺ toxicity towards cells followed by several secondary effects due to a disturbed cellular Ca²⁺ metabolism. Our data illustrate that apparent stimulatory effects of low concentrations of Cd²⁺ on Ca²⁺-dependent enzymes may derive from increased free-Ca²⁺ levels when Cd²⁺ supersedes Ca²⁺ on the ligands.     

The role of chemical speciation,chemical fractionation and calcium disruption in manganese-induced developmental toxicity in zebrafish (Danio rerio) embryos

Manganese (Mn) is an essential nutrient that can be toxic in excess concentrations, especially during early development stages. The mechanisms of Mn toxicity is still unclear, and little information is available regarding the role of Mn speciation and fractionation in toxicology. We aimed to investigate the toxic effects of several chemical forms of Mn in embryos of Danio rerio exposed during different development stages, between 2 and 122 h post fertilization. We found a stage-specific increase of lethality associated with hatching and removal of the chorion. Mn(II), ([Mn(H₂O)₆]²⁺) appeared to be the most toxic species to embryos exposed for 48 h, and Mn(II) citrate was most toxic to embryos exposed for 72 and/or 120 h. Manganese toxicity was associated with calcium disruption, manganese speciation and metal fractionation, including bioaccumulation in tissue, granule fractions, organelles and denaturated proteins.     

Calcium transport affinity,ion competition and cholera toxin effects on cytosolic Ca concentration

Summary The physiological relevance of an apparent ionophore activity of cholera toxin towards Ca²⁺ has been examined in several different systems designed to measure affinity, specificity, rates of ion transfer, and effects on intracellular ion concentrations. Half-maximal transfer rates across porcine jejunal brush-border vesicles were obtained at a concentration of 0.20 M Ca²⁺. When examined in the presence of competing ions the transfer process was blocked by very low concentrations of La³⁺ or Cd²⁺. Sr²⁺, Ba²⁺ and Mg²⁺ were relatively inefficient competitors for Ca²⁺ transport mediated by cholera toxin. The relative affinities observed would be compatible with a selectivity for Ca²⁺ transfer at physiological ion concentrations, as well as an inhibition of this ionophore activity by recognized antagonists of cholera toxin such as lanthanum ions. Entry rates of Ca²⁺ into brush-border vesicles exposed to cholera toxin were large enough to accelerate the collapse of a Ca²⁺ gradient generated by endogenous Ca, Mg-ATPase activity. The treatment of isolated jejunal enterocytes with cholera toxin caused a significant elevation in cytosolic Ca²⁺ concentrations as measured by Quin-2 fluorescence. This effect was specifically prevented by prior exposure of the cholera toxin to excess ganglioside G_M1. We conclude that cholera toxin has many of the properties required for promoting transmembranes Ca²⁺ movement in membrane vesicles and appears to be an effective Ca²⁺ ionophore in isolated mammalian cells.     

Use of a Vibrating Electrode to Measure Changes in Calcium Fluxes Across the Cell Membranes of Oxidatively Challenged Aplysia Nerve Cells

A self-referencing and non-invasive Ca²⁺-sensitive vibrating electrode was used to assess the effects of hydrogen peroxide-induced oxidative challenges on the efflux and influx of calcium across the plasma membrane of single nerve cells cultured from abdominal ganglion of Aplysia californica. A reduced net efflux of Ca²⁺ from the cell soma occurred immediately after the addition of hydrogen peroxide (0.0025 mM, 0.005 mM or 0.01 mM) to the culture medium, indicating damage to the cell membrane or Ca²⁺ transport mechanism. There then followed a marked efflux, the extent and duration of which was related to the concentration of hydrogen peroxide used and which may reflect compensatory activity by the Ca²⁺ regulatory mechanisms in the plasmalemma. No morphological changes were observed in cells challenged with 0.0025 mM hydrogen peroxide and the enhanced rate of Ca²⁺ efflux rapidly decreased to pre-exposure values. Sustained and enhanced Ca²⁺ effluxes from those cells exposed to 0.005 mM or 0.01 mM hydrogen peroxide were also consistent with regulatory pumping of Ca²⁺ out of the cell although contraction and blebbing of neurites and swelling of the soma may indicate that a proportion of the efflux arose from release of Ca²⁺ from disrupted intracellular stores. The vibrating electrode is a useful additional technique for the study of the pathogenesis of neurological conditions, as ionic fluxes across single nerve cells exposed to physiologically-relevant concentrations of free radicals can be monitored non-invasively for prolonged periods.     

Calcium and the control of muscle-specific creatine kinase accumulation during skeletal muscle differentiation in vitro.

A polyacrylamide gel separation method for creatine kinase (CPK) isoenzymes is described, and its use to determine muscle-specific CPK (M-CPK) levels in skeletal muscle cultures is illustrated. In cultures in which cell fusion has been prevented by very low Ca²⁺ concentrations, the increases in M-CPK after 96 hr are similar to those in control cultures. Slightly higher concentrations of Ca²⁺, however, inhibit both cell fusion and M-CPK accumulation. As the calcium concentration is gradually increased further, cell fusion is permitted, followed, at even higher Ca²⁺ levels, by M-CPK accumulation. These effects can be obtained both by adding EGTA to the culture medium and by using Ca²⁺-free culture medium and varying the Ca²⁺ concentration directly. The latter method has the advantage that deleterious effects of EGTA on cell attachment and cell numbers do not occur, even at the lowest Ca²⁺ concentrations. By revealing dramatic effects on CPK levels of small changes in external Ca²⁺ concentrations, these observations may resolve conflicting data in the literature on the question of whether cell fusion is a prerequisite for muscle-specific protein synthesis. Possible mechanisms for the two effects of Ca²⁺ on CPK specific activity (permissive at very low, but inhibitory at intermediate, concentrations) are considered, including membrane mediation, mediation by changes in ionized cytoplasmic Ca²⁺ levels, and possible involvement of cyclic nucleotides.     

Neutral organic solute effects on the activity of the plasma membrane Ca2+-ATPase

We have compared effects of dimethylsulfoxide (Me₂SO) and two polyols on the Ca²⁺-ATPase purified from human erythrocytes. As studied under steady-state conditions over a broad solute concentration range and temperature, Me₂SO, glycerol, and xylitol do not inhibit the Ca²⁺-ATPase activity; this is in contrast to numerous other organic solutes that we have investigated. Under specific experimental conditions, Me₂SO (but not glycerol) substantially increases Ca²⁺-ATPase activity, suggesting a possible facilitation of enzyme oligomerization. The activation is more pronounced at low Ca²⁺ concentrations. In contrast to glycerol, Me₂SO shows no protective effect on enzyme structure as assessed by determining residual Ca²⁺-ATPase activity after exposing the enzyme to thermal denaturation at 45°C. Under these conditions several other organic solutes strongly enhance the denaturating effect of temperature. Because of the temperature dependence of its effect on the Ca²⁺-ATPase activity we believe that Me₂SO activates the Ca²⁺-ATPase by indirect water-mediated interactions.